Skip to main content
. 2021 Jun 11;6(3):e10234. doi: 10.1002/btm2.10234

FIGURE 5.

FIGURE 5

CHIP regulates the binding of FOXO3a with the Bim promoter region. (a) WJMSCs transfected with increasing amounts of siFOXO3a were challenged with HG stress, and the expression levels of FOXO3a and Bim were assessed by immunoblotting. (b) Cells transfected with shcontrol or siFOXO3a in the presence and absence of LY294002 (PI3K inhibitor) were subjected to HG for 24 h, and the activation of FOXO3a, p‐FOXO3a, and Bim was analyzed using Western blot assay. (c) WJMSCs transfected with HA‐vector, HA‐CHIP, shcontrol, shCHIP, or siFOXO3a were incubated with HG for 24 h followed by immunoblotting to analyze the HA, FoxO3a, and Bim levels. (d) WJMSCs were transfected with varying concentration of siCHIP (10, 20, 30 nM) in the presence of HG for 24 h. Thereafter, the extracted cell lysates using cytoplasmic and nuclear fractionation kit were immunoblotted. (e) WJMSCs transfected with HA‐CHIP or shCHIP plasmids were challenged with HG for 24 h and ChIP assay was performed to evaluate the FOXO3a interaction with the Bim promoter region. (f) Prediction of Bim promoter region using NCBI database (brown color indicates Bim promoter region) and converted to three‐dimensional structure for molecular interaction with FOXO3a. (g) Docking studies illustrating the molecular interaction between FOXO3a and the Bim promoter region (Bim region is shown in green color and FOXO3a shown in red color). *p < 0.05, **p < 0.01, and ***p < 0.001 shows the significance. CHIP, carboxyl terminus of Hsc70 interacting protein; HG, high glucose; WJMSCs, Wharton's jelly derived mesenchymal stem cells